Sodium mentholate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-15 - 2021-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals No. 23 (2019)

Version / remarks:

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 3.4, 7.5, 16.5, 36.4 and 80 mg/L and a control
- Sampling method: The samples were taken from the biological phase of the study. Quadruplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test. Quadruplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ -20 °C), protected from light, until liquid-liquid extraction and analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test concentration of nominal 80 mg test item/L was prepared by dissolving 89.8 mg test item into 1122.5 mL test water by intense stirring for 2 hours and ultrasonic treatment for 5 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the daphnids (= start of the test).
- Controls: negative control containing test water (Elendt "M4") without addition of test item
- Test concentration separation factor: 2.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations. All test media were clear and colourless throughout the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus), Water flea
- Strain/clone: clone 5
- Age at study initiation: from 3.50 to 20.75 hours old
- Stage and instar at study initiation: neonates
- Method of breeding: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation
of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
- Source: in-house laboratory culture
- Feeding during test: none

ACCLIMATION
- Acclimation period: Not necessary, since the test was performed in the same medium and under same conditions as the culturing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Water Temperature: 19.3 to 20.3 °C at test start; 19.7 to 19.9 °C at test end

pH:

7.3 to 7.7 at test start; 7.4 to 7.6 at test end

Dissolved oxygen:

9.0 to 9.1 mg/L at test start; 8.8 to 8.9 mg/L at test end

Nominal and measured concentrations:

Nominal: 80, 36.4, 16.5, 7.5 and 3.4 mg test item/L and a control
arithmetic mean measured test concentrations: 66.3, 29.4, 13.4, 5.93 and 2.71 mg test item/L and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of approx. 110 mL
- Type: closed
- Material, size, headspace, fill volume: fill volume: as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some millilitre)
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 (according to OECD 202)
- Alkalinity: 0.9 mmol/L
- Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 500 to 530 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae). Any signs of disease or stress or unusual behaviour were documented in the raw data. The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.


RANGE-FINDING STUDY
- Test concentrations: not specified
- Results used to determine the conditions for the definitive study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, 0.125 - 2 mg/L (nominal), September 2020

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

44.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

66.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

29.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

44.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: one test organism in treatment 13.4 mg/L (arith. mean meas.) with agglutinated antennae
- Mortality of control: 0 %
- Immobilisation of control: After 48 hours of exposure no immobilisation of the test animals was observed in the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Limit test: no
- Dose-response test: yes
- ECx: 24h-EC50 = 0.918 mg/L, 48h-EC50 = 0.685 mg/L
- Other: NOEC = 0.5 mg/L, LOEC = 1 mg/L

Reported statistics and error estimates:

According to the guideline, the 24 and 48-hour EC50 was calculated using the geometric mean of the highest concentration causing no immobility (29.4 mg test item/L) and the lowest concentration resulting in 100 % immobility (66.3 mg test item/L). The software used to perform the statistical analysis was ToxRat Professional.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD 202 taking into account the OECD guidance document no. 23 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards daphnia as representative aquatic invertebrates.
Young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part by GC-MS. Based on the arithmetic mean measured concentrations of the test item the 48-hour NOEC was determined to be 29.4 mg test item/L. The 48-hour LOEC was determined to be 66.3 mg test item/L and the 48-hour EC50 value was determined to be 44.2 mg test item/L, both values also based on arithmetic mean measured test concentrations.

Executive summary:

The toxic effect of the registered substance to Daphnia magna was assessed in a static concentration-response test according to OECD TG 202 taking into account the guidance document no. 23. Young daphnids, less than 24 hours old, from in-house laboratory culture were exposed to 5 test concentrations and a control in groups of 5 individuals per test vessel. Four replicates were prepared for each concentration and the control. To avoid loss of test item due to volatilisation, the test vessels were kept closed during the exposure period of 48 hours. The test conditions (water temperature, dissolved oxygen content, pH and light) were within the ranges requested by OECD TG 202 and the validity criteria stated in the guideline were met.

The biological results were based on the endpoint immobility which was assessed every 24 hours. Besides the highest concentration with 100% immobility, immobility was detected only in the test concentration of 13.4 mg/L (10% immobility). At the lower test concentrations and the control no immobility or other sign of intoxication were observed.

The initial concentrations and the maintenance of the exposure concentrations during the test were analytically verified by GC-MS. The quantification of the test item was performed indirectly by analysing the hydrolysis product menthol in the test samples using liquid/liquid extraction followed by measurement via gas chromatography with MS detection. Direct analysis of the test material was not feasible due to its hydrolysis instability (DT50< 5min). Since the test item concentrations were not within ± 20% of the nominal concentrations during the test, the biological results refer to arithmetic mean measured concentrations.

Based on the arithmetic mean measured concentrations of the test item the 48-hour NOEC was determined to be 29.4 mg test item/L. The 48-hour LOEC was determined to be 66.3 mg test item/L and the 48-hour EC50 value was determined to be 44.2 mg test item/L, both values also based on arithmetic mean measured test concentrations.

Description of key information

1) Key_Short-term toxicity to aquatic invertebrates: EC₅₀(48h) = 44.2 mg/L and NOEC (48h) = 29.4 mg/L based on arithmetic mean measured test concentrations for Daphnia magna (static, freshwater, OECD 202, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

44.2 mg/L

Additional information

The toxic effect of the test item Sodium mentholate to Daphnia magna was assessed in a static concentration-response test according to OECD TG 202 taking into account the guidance document no. 23. Young daphnids, less than 24 hours old, from in-house laboratory culture were exposed to 5 test concentrations and a control in groups of 5 individuals per test vessel. Four replicates were prepared for each concentration and the control. To avoid loss of test item due to volatilisation, the test vessels were kept closed during the exposure period of 48 hours. The test conditions (water temperature, dissolved oxygen content, pH and light) were within the ranges requested by OECD TG 202 and the validity criteria stated in the guideline were met.

The biological results were based on the endpoint immobility which was assessed every 24 hours. Besides the highest concentration with 100% immobility, immobility was detected only in the test concentration of 13.4 mg/L (10% immobility). At the lower test concentrations and the control no immobility or other sign of intoxication were observed.

The initial concentrations and the maintenance of the exposure concentrations during the test were analytically verified by GC-MS.The quantification of the test item Sodium mentholate was performed indirectly by analysing the hydrolysis product menthol in the test samples using liquid/liquid extraction followed by measurement via gas chromatography with MS detection. Direct analysis of Sodium mentholate was not feasible due to its hydrolysis instability (DT₅₀< 5min). Since the test item concentrations were not within ± 20% of the nominal concentrations during the test, the biological results refer to arithmetic mean measured concentrations.

Based on the arithmetic mean measured concentrations of the test item the 48-hour NOEC was determined to be 29.4 mg test item/L. The 48-hour LOEC was determined to be 66.3 mg test item/L and the 48-hour EC₅₀ value was determined to be 44.2 mg test item/L,both values also based on arithmetic mean measured test concentrations.